Heat-Shock Proteins Associated with Base Excision Repair Enzymes in HeLa Cells

Abstract

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

“…Stimulation of HAP1 and ␤ pol to eliminate abasic sites might well account for this protective effect, but other mechanisms of Hsp70 stimulation are still not excluded. Repair of DNA damage in THP-1 cells after ionizing radiation was also stimulated by Hsp70, presumably after stimulation of ␤ pol repair of strand breaks (Mendez et al 2000). Pretreatment of THP-1 cells by Hsp70 for 3 hours enhanced the size of the shoulder in dose-response curves (Fig 5).…”

“…Previous publications demonstrated specific attachment and strong stimulation of DNA BER enzymes by Hsp70 in vitro (Mendez et al 2000(Mendez et al , 2003a(Mendez et al , 2003bKenny et al 2001). We reasoned that cell viability (clonogenic fraction) might be protected by the enhanced BER to be expected in Hsp70-treated cells.…”

Over-expressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

“…Ciavarra et al (1994) reported that nuclear type I topoisomerase complexes with Hsc70 might limit heat-induced protein damage, accelerate restoration of protein function in an ATPindependent reaction, or both. Some studies showed that Hsp70 can improve base excision repair by interacting with human apurinic/apyrimidinic endonuclease and by stimulating single-strand gap-filling by DNA polymerase beta (Mendez et al 2000(Mendez et al , 2003Kenny et al 2001;Bases 2005). In a previous study, we found a significantly negative correlation between Hsp70 levels and the level of DNA damage as measured by the comet assay in workers exposed to coke oven emissions (Xiao et al 2002).…”

“…Taking this into account, we can speculate that Hsp70 could be involved in the signaling of protein synthesis in the p53-DNA damage pathway that modulates DNA repair. In addition, Hsp70 might act at the DNA repair level because this Hsp has been shown to interact with the base excision repair enzymes (Mendez et al 2000;Kenny et al 2001) and stimulate DNA polymerase beta gap-filling activity (Mendez et al 2003).…”

Modulation of GdCl3and Angelica Sinensis polysaccharides on differentially expressed genes in liver of hepatic immunological injury mice by cDNA microarray

“…Krokan et al propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution [23] . But Kvaløy et al find mutations affecting the function of human UNG gene are seemingly infrequent in human tumor cell lines [26,27] . So we infer down-regulation of these genes may affect cell proliferation, and GdCl 3 and ASP protect liver depend on this m ec h an i sm in s o me de g re e .…”